Hydrolysis is a chemical process in which a water molecule is added to a substance resulting in the split of that substance into two parts. One fragment of the target molecule (or parent molecule) gains a hydrogen ion (H+) from the split water molecule. The other portion of the target molecule collects the hydroxyl group (OH—) of the split water molecule. In effect an acid and a base are formed.
There are many known compounds which are susceptible to hydrolysis leading to significant degradation of the compounds. Degradation due to hydrolysis leads to difficulty in commercial use of such compounds due to their difficulty in formulating and limiting the impurities under the regulatory specification hence hydrolysis plays an important role in formulating or developing any compound for commercial use.
Azacitidine and Decitabine are chemical analogues of cytidine, a nucleoside present in DNA and RNA. Azacitidine and its deoxy derivative, Decitabine (also known as 5-aza-2′ deoxycytidine), are used in the treatment of Myelodysplastic Syndrome. Both drugs were first synthesized in Czechoslovakia as potential chemotherapeutic agents for cancer. Azacitidine belongs to the class of antimetabolites that bear structural similarity to naturally occurring substance, of significance is Cytidine analogs is a pyrimidine nucleosides, generically known as Azacitidine and Decitabine represented by Formula-I & Formula-II respectively, these are antineoplastic agents indicated for the treatment of myelodysplastic syndrome. These analogs of Cytidine have demonstrated significant inhibitory effects on tumor growth in both in vitro and in vivo models.

Azacitidine acts as a false substrate and potent inhibitor of methyltransferases leading to reduction of DNA methylation affecting the way cell regulation proteins are able to bind to the DNA/RNA substrate However Decitabine is hypomethylating agent, it hypomethylates DNA by inhibiting methyltransferase.
Presently, Azacitidine and Decitabine are marketed as an injectable formulation under the brand name VIDAZA™ and DACOGEN™ respectively, which are available as a sterile lyophilized powder.
Another compound of significance which is susceptible to hydrolytic degradation is Bendamustine chemically known as (4-{5-[Bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl}butyric acid, is an atypical structure with a benzimidazole ring, whose structure includes an active nitrogen mustard (see Formula III, which shows Bendamustine Hydrochloride).

Because of their high reactivity in aqueous solutions, nitrogen mustards are difficult to formulate as pharmaceuticals and are often supplied for administration in a lyophilized form that requires reconstitution, usually in water, by skilled hospital personal prior to administration. Once in aqueous solution, nitrogen mustards are subject to degradation by hydrolysis, thus, the reconstituted product should be administered to a patient as soon as possible after its reconstitution.
Bendamustine was initially synthesized in 1963 in the German Democratic Republic (GDR) and was available from 1971 to 1992 in that location under the name Cytostasan®. Since that time, it has been marketed in Germany under the tradename Ribomustin®. It has been widely used in Germany to treat chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and breast cancer.
Bendamustine Hydrochloride is marketed as TREANDA® for Injection is an alkylating drug which is indicated for treatment of patients with Chronic Lymphocytic Leukemia (CLL) and Indolent B-cell non-Hodgkin's lymphoma (NHL) that has progressed during or within six months of treatment with Rituximab or a Rituximab containing regimen.
Due to its degradation in aqueous solutions (like other nitrogen mustards), Bendamustine is supplied as a lyophilized product. The current lyophilized formulation of Bendamustine (Ribomustin®) contains Bendamustine Hydrochloride and Mannitol in a sterile lyophilized powder. The finished lyophilisate is unstable when exposed to light. Therefore, the product is stored in brown or amber-colored glass bottles.
U.S. Pat. No. 4,684,630 discloses a method of parenterally delivering the aqueous-unstable 5-azacytosine arabinoside and 5-Azacitidine compound involving an aqueous dilution of a stable, anhydrous organic solution having the drug dissolved therein. The resulting organic aqueous solution is physiologically suitable for parenteral delivery into warm blooded mammal and contains the drug in an effective dosage concentration per unit volume. The patent discloses the methods of diluting the aqueous unstable anticancer agent with the combination of the aqueous and organic solvents where the preferred organic solvents are Dimethylsulfoxide and Dimethylacetamide.
U.S. Patent Application No. 2006/0159713 relates to the pharmaceutical compositions of lyophilized Bendamustine suitable for pharmaceutical use. According to this patent Application the stability of the Bendamustine is low in aqueous environment hence it has to be administered as soon as possible after reconstitution. This Patent Application reveals the Lyophilized pharmaceutical compositions and discloses the various solvents that can be employed for lyophilization which includes tertiary butanol, n-propanol, n-butanol, isopropanol, ethanol, methanol, acetone, ethyl acetate, dimethyl carbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone, 1-pentanol, methyl acetate, carbon tetrachloride, dimethyl sulfoxide, hexafluoroacetone, chlorobutanol, dimethyl sulfone, acetic acid, and cyclohexane. Preferred organic solvents include one or more of ethanol, methanol, propanol, butanol, isopropanol, and tertiary butanol. A more preferred organic solvent is tertiary butanol.
U.S. Patent Application No. 2005/0020615 relates to the Lyophilized CCI-779 formulations composed of CCI-779 and t-butyl alcohol or ethanol.
PCT Application No. WO2011014541 relates to Azacitidine containing compositions that are stable for at least about 24 hours at room temperature, i.e. temperatures of less than or equal to about 25° C. The Patent Application discloses kits containing an amount of Azacitidine or a pharmaceutically acceptable salt thereof, preferably in lyophilized form, in a first vial or container and a pharmacologically suitable fluid which contains at least one of Propylene glycol or Poly Ethylene Glycol, or mixtures thereof and an effective amount of a surfactant, i.e. at least about 0.5%, in a second vial or container. The invention uses the pharmacologically suitable fluids like Propylene glycol or Poly Ethylene Glycol and an effective amount of surfactant and provided as a kit along with lyophilized Azacitidine
U.S. Patent Application No. 2011/0042247 relates to the pharmaceutical formulations for parenteral administration comprising Azacitidine or its pharmaceutically acceptable salts and processes for preparing the formulations which includes Lyophilization process. According to the invention the formulation contains the mannitol and water as the ingredients the aqueous solution is prepared at about −3° C. to about −1° C. and then lyophilizing the solution the solvent used here is water and mannitol is used as bulking agent.
The literature reports that VIDAZA™, reconstituted with 4 mL of sterile water for injection to form a suspension for subcutaneous administration, may be stored for up to 1 hour at 25° C. or for up to 8 hours between 2° C. and 8° C. VIDAZA™ reconstituted with 10 mL of sterile water for injection for intravenous administration may be stored at 25° C., but the administration must be completed within 1 hour after reconstitution. The duration of IV infusion administration is limited by the decomposition and instability of Azacitidine, and low aqueous solubility of the drug in aqueous solutions. Further Azacitidine hydrolyzes quickly in water, converting into other forms.
A commercially available product containing Decitabine is sold as Dacogen® by MGI Pharma. The Dacogen® product is for injection and is available as white to almost white lyophilized sterile powder supplied in glass vial. Each 20 mL single dose glass vial contains Decitabine, monobasic potassium phosphate, and sodium hydroxide. Each Dacogen™ vial has to be aseptically reconstituted with 10 mL of sterile water for injection, and upon reconstitution each mL contains approximately 5 mg of Decitabine at pH 6.7-7.3. Immediately after reconstitution, the solution should be further diluted with 0.9% of Sodium Chloride injection, 5% Dextrose Injection, or lactated Ringer injection, to a final concentration of 0.1-1. mg/mL.
The reconstitution of the present commercially available Bendamustine lyophilized powder is difficult. Reports from the clinic indicate that reconstitution can require at least fifteen minutes and may require as long as thirty minutes. Besides being burdensome and time-consuming for the healthcare professional responsible for reconstituting the product, the lengthy exposure of Bendamustine to water during the reconstitution process increases the potential for loss of potency and impurity formation due to the hydrolysis of the product by water.
The manufacture of a freeze dried product necessitates that the product is usually first manufactured as a solution, filtered to sterilize, aseptically filled, and finally lyophilized to remove the solvents. All of these unit operations require that the product be held in the solution state for a defined period of time, at lease for 4 hours. Fabrication of the compounds of the invention in aqueous solution leads to the extensive degradation at 5±3° C.
There exists a need to develop the process of preparing the dosage form of such compounds which can overcome the limitations of the existing processes of manufacturing the finished dosage form such as degradation due to hydrolysis which is the most important aspect of any formulation. The present invention provides advantages in preventing the degradation of the compounds which are susceptible to hydrolysis by using the mixture of aqueous and organic solvent in a particular proportion to depress the freezing point of the solution to a certain level, hence enabling to work at subzero temperatures by maintaining the solubility at that temperature. None of the prior art documents mention the use of the mixture of aqueous and organic solvent to work at subzero temperature i.e. −5±2° C. By using the appropriate combination of the solvents and their concentration degradation of the prelyophized solution can be controlled up to good extent. It was surprisingly found that this process provides the compositions with better degradation profile than the processes for preparation of the compositions for hydrolysis susceptible compounds already known.